Contents

Materials

Glassware & Equipment

Falcon tubes

500μl Eppendorf tubes, on ice

200ml conical flask

200μl pipetman or repeating pipettor

5ml pipette

Preparation

Grow a 5ml overnight culture of cells in LB media. In the morning, dilute this culture back into 25-50ml of fresh LB media in a 200ml conical flask. You should aim to dilute the overnight culture by at least 1/100.

Grow the diluted culture to an OD600 of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD600 0.2)

Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is X ml, you will need X tubes. At this point you should also make sure that your TSS is being chilled (it should be stored at Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle 4^o}
C but if you have just made it fresh then put it in an ice bath).

Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.

All subsequent steps should be carried out at Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle 4^o}
C and the cells should be kept on ice wherever possible

Remove supernatant. The cell pellets should be sufficiently solid that you can just pour off the supernatant if you are careful. Pipette out any remaining media.

Resuspend in chilled TSS buffer. The volume of TSS to use is 10% of the culture volume that you spun down. You may need to vortex gently to fully resuspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall.

The original paper [1] suggests freezing the cells immediately using a dry ice bath. I (BC) have used liquid nitrogen quite successfully instead of dry ice. Simply placing the cells at Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle -80^o}
C also seems to work well (Jkm)

If you run a control every time you clone (i.e. a vector-only ligation), you can as well freeze cells in 200 μl aliquots. Unused cells can be frozen back once and reused, albeit with some loss of competence.

It is a good idea to run a positive control on the cells.

The Endy Lab is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out here.

Notes

Note1: CT Chung paper recommends long storage at -70˚C, while people have been using a wide range of temperatures from -20˚C to -140˚C for long term storage.